The discussion in this section is not limited to subject matter that qualifies as “prior art” against the present invention. Therefore, no admission of such prior art status shall be implied or inferred by reason of inclusion of particular subject matter in this discussion, and no declaration against the present inventors' interests shall be implied by reason of such inclusion.
Autoimmune Diseases
Autoimmune diseases result from the loss of tolerance to certain self antigens, resulting in an inappropriate attack by the immune system upon these antigens. Numerous mechanisms normally function to maintain immune self-tolerance in both the antibody-mediated (humoral) and cellular aspects of the immune system. It is when these mechanisms malfunction that autoimmune diseases occur.
Illnesses resulting from such misdirected immune system activity affect more than 10 million Americans. Therapies that treat the causes, rather than the symptoms of these diseases have long been sought. While various agents have been found that provide beneficial reductions in autoimmune activity, such treatments, in general, have the undesirable and dangerous effect of also compromising normal immune functions, and are thus considered sub optimal.
Multiple Sclerosis
Multiple Sclerosis (MS) is a progressive neurodegenerative autoimmune disorder affecting about 350,000 Americans. Females are twice as likely as males to develop the disease. MS usually affects patients who are between the ages of 15 and 50 years, most commonly young women between the ages of 20 and 40. MS derives its name from the multiple scarred (sclerotic) areas of degeneration visible on macroscopic examination of the central nervous system (CNS) of affected individuals. The degeneration associated with MS includes demyelination, chronic inflammation, and gliosis (scarring) of affected areas of the brain, optic nerve, and spinal cord.
MS is characterized by different types and stages of disease progression. Patients are diagnosed as having relapsing and remitting MS when they experience periods of exacerbations and remissions. Rapidly progressive or chronically progressive MS is diagnosed depending upon the pace of disease progression. These stages usually occur later in the course of the disease when the extent of recovery from individual attacks decreases and there are clinically stable periods between periods of deterioration. Inactive MS typically occurs late in disease progression and is characterized by fixed neurologic deficits of variable magnitude.
MS is always debilitating and may sometimes lead to paralysis and death. Although the factors triggering the initial onset of MS remain unknown, evidence is persuasive that MS pathology results from the autoimmune actions of certain white blood cells, especially neuroantigen-specific T cells.
Pathologically, MS is characterized by chronic inflammation, demyelination, and gliosis of white matter. The classic lesions of MS, termed plaques, are well-demarcated gray or pink areas easily distinguished from surrounding white matter. (The coloration of white matter is due to the high concentrations of myelin in this tissue.) The acute MS lesion is characterized by demyelination associated with tissue infiltration by mononuclear cells, predominantly T cells (both helper and cytotoxic) and macrophages, with B cells and plasma cells rarely being present. These inflammatory infiltrates appear to mediate the demyelination that is characteristic of the disease. Since activated T cells release cytokines that promote macrophage infiltration and activation, T cells are considered the primary mediators of pathogenic autoimmune attack in MS. More detailed discussions of T cells and myelin are found below under “T Cell Physiology,” “T Cells and Autoimmune Pathogenesis,” and “T Cells Target Defined Autoantigens in MS.”
Current treatments for MS vary. Depending on the severity of disease and the response to treatment, a variety of options for drug therapy are available. Drugs used to treat MS include steroids such as prednisone and methylprednisolone, hormones such as adrenocorticotropic hormone (ACTH), antimetabolites such as azathioprine, alkylating agents such as cyclophosphamide, and T-cell inhibitory agents such as cyclosporine. The administration of any of these drugs is dangerous, as they all typically produce some level of generalized immunosuppression and leave the patient more prone to infection. Patients may also experience side effects such as nausea, hair loss, hypertension, and renal dysfunction when treated with such drugs. In addition, some of these drugs are carcinogenic.
New approaches to treating MS include interferon-beta therapy, which can lessen the frequency of MS attacks and slow disease progression. Other new approaches include administration of antigens involved in MS autoimmune responses, as discussed below.
Diagnosis of MS
MS is typically diagnosed based on medical history and physical examinations. No clinical signs or diagnostic tests are unique to MS. Diagnosis of a patient with a single, initial symptom commonly associated with MS cannot be definitive, although symptoms of relapsing and remitting disease increases the likelihood of an MS diagnosis. Two or more episodes of worsening each lasting 24 hours or occurring at least a month apart, or slow stepwise progression of signs and symptoms over at least six months are considered strong indications of MS. MRI findings implicating involvement in two or more areas of CNS white matter and evidence of systemic disease are also indicative of MS.
Currently, various laboratory tests are performed to confirm the diagnosis and assess the progression of the disease. Such tests include analysis of human cerebrospinal fluid (CSF) and blood for chemical and cellular signs of MS pathology.
CSF abnormalities associated with MS consist of mononuclear cell pleocytosis and the presence of autoreactive (typically myelin reactive) T cells, an elevation in the level of total Ig, and the presence of oligoclonal Ig, typically seen as two or more oligoclonal bands. In approximately 80 percent of patients, the CSF content of IgG is increased in the presence of a normal concentration of total protein. This results from the selective production of IgG within the CNS.
Oligoclonal banding of CSF IgG is detected by agarose gel electrophoresis techniques. Two or more oligoclonal bands are found in 75 to 90 percent of MS patients. The presence of oligoclonal banding correlates with an elevated total IgG level in MS. Other Ig abnormalities in MS CSF include free kappa or lambda light chains and elevated levels of other Ig isotypes including IgA.
Metabolites derived from myelin breakdown also may be detected in CSF. Elevated levels of MBP or its fragments may be detected, e.g., by radioimmunoassay, both in MS and in some patients with other neurologic diseases.
In addition to many of the pathologic signs described above for CSF, blood of MS patients may show increased levels of IgG synthesis, polymorphonuclear leukocytes, decreased serum B12 levels, elevated erythrocyte sedimentation rate, and presence of autoantibodies or autoreactive T cells. As discussed below, the “reactive T cell index” is a particularly useful cellular finding for monitoring the clinical course of MS.
While these various indicators of MS disease are clinically useful, other means of following the course and extent of autoimmune activity in MS patients using relatively inexpensive and easily quantifiable tests, such as blood or cerebrospinal fluid tests (as opposed to expensive imaging techniques such as MRI) are needed.
T Cells, Antigen Presenting Cells, and T Cell Epitopes
As mentioned above, MS pathogenesis is believed to be mediated by the inappropriate actions of white blood cells (leukocytes), most importantly T cells. T cells are mononuclear white blood cells that provide many essential immune functions. The importance of T cells in human autoimmune diseases has been increasingly appreciated in the past decade. Studies using treatments that result in generalized immunosuppression have defined a critical role for a subset of T cells, known as CD4+ or helper T cells, as primary regulators of all immune responses (both cellular and humoral) to protein or peptide antigens.
T cells mediate tissue injury by indirect and direct means. T cells of both CD8+ (cytotoxic) and CD4+ (helper) subsets secrete a variety of inflammatory cytokines that can damage tissues indirectly by activating various other types of white blood cells. Examples of such T cell effects include activation of antibody secreting B cells (stimulating humoral immune activity) and activation of macrophages, which can cause acute tissue damage and inflammation by releasing hydrolytic enzymes, reactive oxygen species, and additional pro-inflammatory cytokines. In addition to these indirect effects of T cell activity, direct tissue damage can be mediated by CD8+ cytotoxic T cells attacking cells displaying target antigens.
One unique aspect of the physiology of T cells is the presence of membrane bound antibody-like binding structures called T cell receptors (TCRs) on their cell surfaces. Like antibodies, TCRs bind with high specificity to particular antigens. Like antibody-producing cells, which develop as multitudinous clones of cells, each clone producing antibodies with unique specificities, T cells develop as a vast number of distinct clones, and any particular T cell clone expresses a single type of TCR with a defined binding specificity. T cell clones with TCRs that bind specifically to self antigens are responsible for the development of autoimmune diseases.
In addition to being cell surface, rather than soluble molecules, TCRs differ from antibodies in the way they recognize antigens. While antibodies bind to antigens in various contexts (e.g., antigens that are native, denatured, soluble, or membrane bound), TCRs only bind to most antigens after the antigens have been broken down (processed) by certain cells known as antigen presenting cells (APCs) and the resulting peptides displayed (presented) on the cell surfaces of the APCs in association with class II or class I proteins of the major histocompatibility complex (MHC). The details of the mechanism by which antigen processing is carried out by APCs are poorly understood. There is consequently considerable uncertainty regarding the ability of APCs to process a given antigen in such a way as to produce and display a particular peptide unless that antigen has already been characterized in this respect.
One exception to the requirement that APCs process and present antigens in order for the antigens to stimulate T cells via their TCRs is the case of small peptide antigens. Such peptides can bind directly to MHC class I molecules on cell surfaces without being processed by APCs, and may then be “recognized” and bound by specific TCRs and thereby stimulate T cells.
Studies of the interactions of antibodies and TCRs with their specific antigens have shown that a particular polypeptide antigen typically comprises numerous submolecular features, known as epitopes, that each can serve as a distinct binding site for a particular antibody or (subsequent to APC processing of the polypeptide and MHC display of a derived peptide comprising the T cell epitope) a particular TCR.
Thus, TCRs and antibodies are similar in that each only recognizes a small portion of a polypeptide antigen. They differ in that an antibody typically recognizes its specific epitope within the context of the intact polypeptide, while a TCR only recognizes a specific epitope as an MHC class II or class I associated peptide fragment of a processed polypeptide on the surface of an APC. Importantly, this TCR epitope recognition process can only occur if an APC can process the polypeptide antigen so as to generate and display the appropriate peptide. Thus, even though a peptide that is recognized by a specific TCR may be present in a particular polypeptide antigen, it is uncertain whether peptides capable of stimulating T cells expressing that specific TCR will be derived from that polypeptide antigen in vivo. This is because it is uncertain whether APCs can generate the peptide recognized by the specific TCR by processing the particular polypeptide antigen.
This lack of certainty regarding the results of APC processing of a particular polypeptide antigen stems from several factors. One reason why an APC may not process a particular polypeptide antigen so as to display a specific peptide epitope contained within the polypeptide is that the APC efficiently cleaves the polypeptide at a site within the epitope and thereby destroys it. A second reason is that the polypeptide cannot enter into or be effectively broken down by the subcellular compartments of APCs responsible for polypeptide processing.
Certain aspects of the primary structure (linear amino acid sequence), secondary structure (3D structure resulting from interactions of amino acid residues that are close to one another in the linear amino acid sequence), or tertiary structure (3D structure resulting from interactions of amino acid residues that are far from one another in the linear amino acid sequence but come into proximity with each other as a result of folding of the polypeptide chain) can impact APC processing. The amino acid sequence of a polypeptide is clearly the most important factor in determining its potential to be processed and displayed by APCs so as to stimulate specific T cells. The peptide recognized by the specific T cell's TCRs must be contained within the amino acid sequence of the polypeptide. The amino acid sequence also determines the potential secondary and tertiary structure (i.e., the folding) of the polypeptide.
The folding of a polypeptide can also significantly impact APC processing. Both the first and second reasons given above for the uncertainty of the display by APCs of a specific epitope derived from a particular polypeptide can result from the way in which the polypeptide is folded. Proteolytic cleavage during processing within the APC can be influenced by the exposure or masking of a cleavage site due to folding. Entry of polypeptides into subcellular compartments is well known to be influenced by the 3D structure of the polypeptide, which structure is a function of folding.
T Cells and Autoimmune Diseases
In autoimmune diseases, only a limited number of T cell clones, reactive with various epitopes of a small number of autoantigens, become activated and are involved in pathogenesis. Various mechanisms have been postulated to play a role in this pathogenic activation of disease-causing autoreactive T cells. Primary activation of antigen presenting cells (APCs) by infection or local inflammation is implicated in one such mechanism. APCs activated in this way can then provide powerful co-stimulation for hitherto unreactive T cells.
Other proposed mechanisms involve the polyclonal activation of previously quiescent autoreactive T cells by superantigens, such as bacterial toxins; or a coincidental molecular mimicry between foreign and self antigens (Abbas et. al. 1994). In this last case, the host immune system mounts a response to an epitope on a protein expressed by a pathogen, such as a virus, that resembles a homologous epitope on a host protein. Autoimmune attack then results from the cross-reactive immune response that ensues. In addition to external factors, underlying the emergence of all T cell-mediated autoimmune disease is a complex pattern of inherited susceptibility determined by multigenic factors.
In several autoimmune diseases, including MS (as discussed in detail immediately below under “T Cells Target Defined Autoantigens in MS”), some or all of the autoantigens targeted by pathogenic T cells have been identified. Knowledge of these self antigens and the specific epitopes within these antigens that are targeted by autoreactive T cells in an autoimmune disorder such as MS provides an approach to therapy, as discussed in detail below under “Treatment of MS by Administration of Antigens” and “Therapeutic Induction of Apoptosis”.
T Cells Target Defined Autoantigens in MS
Although, as discussed above, the precise etiology of MS remains unknown, autoimmune attack is clearly responsible for the destruction of central nervous system (CNS) myelin that is the hallmark of the disease. Myelin is the characteristic component of the myelin sheath that surrounds the axons of certain neurons, acts as an electrical insulator, and is essential for the proper signal transmission functions of these neurons. The demyelination associated with MS thus causes a loss of function in affected neurons, disrupting neuronal signaling and leading to paralysis and severe impairment of sensory functions.
The myelin sheath is made by oligodendrocytes (in the central nervous system) and Schwann cells (in the peripheral nervous system). Myelin is composed of regularly alternating layers of lipids (e.g., cholesterol, phospholipids, and sphingolipids) and proteins.
The four major protein components of myelin, i.e., myelin basic protein (MBP), proteolipid protein (PLP), myelin associated glycoprotein (MAG) and myelin oligodendrocyte protein (MOG), are recognized by autoreactive T lymphocytes isolated from MS patients (Martin et al. 1992; Kerlero de Rosbo et al. 1993).
Myelin basic protein (MBP) is a major protein component of myelin, comprising approximately 30% of the total protein content of the myelin sheath. MBP has been shown to be a major target autoantigen in MS, and T cells reactive with MBP play a key role in its pathogenesis (see, for example, Schwartz, R S, “Autoimmunity and Autoimmune Diseases” in Paul, Fundamental Immunology, 3rd Ed. Raven Press, New York, 1993, pp. 1033–1097; Brown and McFarlin 1981. Lab Invest 45, pp. 278–284; Lehmann et al. 1992. Nature 358, pp. 155–157; Martin et al. 1992. Ann Rev Immunol 10, pp. 153–187; Sprent 1994. Cell 76, pp. 315–322; Su and Sriram. 1991. J of Neuroimmunol 34, pp. 181–190; and Weimbs and Stoffel. 1992. Biochemistry 31, pp. 12289–12296).
MBP-specific T lymphocytes are found in the blood of MS patients. While they can sometimes be found in the blood of healthy individuals, they are typically present in the cerebrospinal fluid (CSF) of patients with MS. Significantly, such T cells are not found in CSF from healthy individuals (Kerlero de Rosbo et al. 1993; Zhang et al. 1994).
The immune responses of MS patients towards MBP clearly differ from those of healthy individuals. MBP reactive T cells are preferentially activated in MS patients, as demonstrated by the observation that the frequency of MBP-specific T cells expressing markers of activation (e.g., IL-2 receptors) is elevated in MS patients (see, for example, zhang, et al., 1994).
Gene mutation frequency analysis also provides evidence that MBP reactive T lymphocytes are specifically activated in MS patients. Since gene mutation is more frequent in dividing than in resting T cells, an increased mutation frequency in T cells of a particular specificity provides an indication of the specific activation of those cells in vivo (Allegretta et al. 1990).
T lymphocytes from MS patients were cultured in thioguanine to test the frequency of mutations in the hprt gene that would render them resistant to this purine analogue. A high frequency of thioguanine resistant T cell clones, up to 10 times the frequency of T cells from normal individuals, was found in MS patients, and a significant percentage of these mutant clones proliferated in response to brain MBP, although they had never been intentionally exposed to this antigen. In contrast, no resistant clones obtained from normal subjects recognized MBP.
MBP is also considered to be a primary autoantigen in MS because of its ability to induce experimental allergic encephalomyelitis (EAE) in animals. EAE is an experimentally induced condition that closely resembles MS and provides the only animal model of MS. In addition, transfer of T cells from an individual suffering from MS or EAE to a healthy animal can produce EAE in the recipient, a method of disease induction referred to as “adoptive transfer”. For example, in human to animal transfer studies, CSF mononuclear cells (including T cells) from MS patients caused paralysis, ataxia, and inflammatory brain lesions when injected into the CSF in the brain ventricles of severe combined immunodeficiency (SCID) mice (Saeki et al. 1992). Also, immunization of animals with MBP can elicit the CNS inflammation, paralysis, and other signs and symptoms of EAE (see, for example, Martin et al. 1990, and Abbas et al. 1994).
Although it is clear that MBP is one of the primary antigens targeted by autoreactive T cells in MS, studies have revealed a marked heterogeneity of MBP epitopes that can induce T cell proliferative responses. These studies have not consistently revealed a single MBP epitope that is recognized with higher frequency by reactive T cells of MS patients than those of normal healthy individuals (Chou et al. 1989; Richert et al. 1989; Martin et al. 1990; Ota et al. 1990; Pette et al. 1990; Martin et al. 1992; Meinl et al. 1993).
Different molecular forms (isoforms) of MBP are generated by differential splicing of MBP hnRNAs, resulting in the presence in the encoded protein of some or all of the seven exons of the single MBP gene. In healthy adults, MBP is found almost exclusively as an 18.5 kDa molecule which is produced from an mRNA comprising all exons of the MBP gene except exon 2 (Kamholtz et al. 1988). Other forms of MBP include a full length (all 7 exons) 21.5 kDa isoform, and two other minor isoforms (17.2 and 20.2 kDa). The expression of the two exon 2 containing isoforms (21.5 kDa and 20.2 kDa) appears to increase with myelin formation, during both early fetal development and remyelination of damaged tissue (Kamholtz et al. 1988; Roth et al. 1987). These two isoforms are referred to in the art, and herein, as “fetal” isoforms, although they are also found in remyelinating damaged adult tissue.
MS plaques contain areas of remyelination and thus should contain higher levels of the 21.5 isoform of MBP than found in healthy adult CNS tissue, suggesting that an immune response to an epitope within the common 26 amino acid region (corresponding to the sequence spanning amino acid residue 60 to amino acid residue 85 of SEQ ID NO:1) of each of the two fetal isoforms of MBP coded for by exon 2 (which regions are referred to as “X2MBP” or simply “X2”) could exacerbate the clinical course of established disease (Prineas et al. 1993; Raine and Wu, 1993; Bruck et al. 1994).
Since remyelination may occur cyclically in the course of MS, each cycle of remyelination could theoretically serve to drive an ongoing immune response by activating resting X2MBP specific T cells in the CNS. Supporting this hypothesis, several lines of evidence suggest the involvement of an epitope encoded by exon 2 of the MBP gene (i.e., an epitope within X2MBP) in MS pathogenesis.
Studies of the role of alternate isoforms of MBP in MS require the availability of quantities of purified myelin antigens in order to evaluate their immunological properties. Such studies have therefore generally been limited to utilizing synthetically-derived peptides, e.g., peptides comprising X2MBP. Recently, CD4+ MHC class II-restricted T cells reactive with peptides containing exon 2 encoded sequences of human MBP were isolated from peripheral blood of both MS patients and normal healthy controls (Voskuhl et al., 1993a; Voskuhl et al. 1994). In a family afflicted with MS, the frequency of T lymphocytes specific for an X2 comprising peptide was higher than the frequency of T cells specific for epitopes within the 18.5 kDa isoform of MBP that does not contain X2 (Voskuhl et al., 1993b). In addition to this data from human subjects, a murine X2 comprising peptide was recently found to be immunogenic in SJL/J mice, and severe EAE was induced by adoptive transfer of exon 2 peptide-sensitized lymphocytes (Segal et al., 1994; Fritz and Zhao, 1994).
Taken together, these human and animal findings demonstrate that an in vivo cellular immune response to the myelin derived antigen MBP causes at least some of the pathogenesis associated with multiple sclerosis. It should be noted, however, that all of the studies regarding X2 epitopes used synthetic peptides as antigens and none of them used full length MBP 21.5 protein. In light of the uncertainty regarding processing and display of particular epitopes of untested proteins by APCs, it has been questioned in the art whether these results are truly relevant to in vivo MS pathogenesis.
Treatment of MS by Administration of Antigens
The ideal therapeutic treatment for any disease is one that specifically blocks pathogenesis without affecting normal physiology. In the case of autoimmune diseases, an approach to such ideal therapy is a treatment that specifically induces immune tolerance to autoimmune disease-associated self antigens without affecting immune responses to foreign antigens. New therapeutic agents and treatment strategies are being sought that will allow the induction of tolerance to specific autoantigens, while leaving all other aspects of immune function unaltered. Attempts have been made to therapeutically modify T cell responses via the administration of antigens to suppress specific autoreactive T cells and thereby elicit tolerance to disease-associated autoantigens. A distinct advantage of such antigen-specific therapy is that it can achieve the therapeutic modulation of the activities of only those T cells that, by reacting with the self antigens, are responsible for the development of pathology. This specificity provides therapeutic benefits without altering the important immune activities of T cells reactive with other antigens.
MS antigens have been studied as tolerization inducers for the treatment of MS/EAE, since therapies that suppress autoreactive T cells may significantly alleviate nervous tissue demyelination and resulting symptoms (see, for example, Adronni et al. 1993 and Critchfield et al. 1994). A number of treatment protocols and antigens have been used in these studies, with animal rather than human forms of the antigens predominantly being used. For example, Weiner et al. Science 1993, 259:1321–1324, used MBP purified from bovine myelin and Miller et al. 1992 used guinea pig, rat, and mouse MBPS. In studies using human antigen, MBP was purified from cadaveric human brain (See, for example, Zhang, et al. 1994).
Oral tolerance involves regulatory CD8+ T cells that suppress immune responses both in vitro and in vivo through the secretion of cytokines, including TGF-beta (Chen et al. Science 1994, 265:1237–1240). The down-regulation of the activity of T cells mediated by this mechanism is not specific to particular T cell clones, and does not involve the production of antigen-specific suppressor factors, but acts on any T cells in close enough proximity to the suppressive T cells to be affected by their secreted cytokines.
Recent studies have investigated the tolerizing effects of oral administration of bovine myelin to MS patients (Weiner et al. 1993 Science 259:1321–1324; Yoon, 1993 Science 259:1263). While fewer of the patients treated with oral myelin developed exacerbations of their MS symptoms than the patients treated with placebo, the results of the study were inconclusive, as the patients were not properly randomized. In fact, the authors cautioned that “It must be strongly emphasized that this study does not demonstrate efficacy of oral myelin in the treatment of MS.” Thus, while oral tolerization studies support the usefulness of myelin proteins as immunomodulatory agents for the treatment of MS, new, more effective antigens, and alternative modes of administration of such antigens for the immunomodulatory treatment of MS continue to be sought.
Clearly, for the treatment of human disease, human-derived antigens have advantages over animal-derived antigens, as they are the actual autoantigens targeted for autoimmune attack in human disease, and suppression of disease should be most effective when homologous protein is administered (Miller et al. 1992). In fact, it is known that immunodominant epitopes (i.e. the antigenic regions of the protein most often recognized by CD4+ autoreactive T cells) of important MS autoantigens differ depending on the species from which the antigen is derived, even though many myelin antigens exhibit high interspecies homology at the amino acid sequence level. For example, as determined by analysis of T cells obtained from MS patients, an immunodominant epitope of human MBP is contained with the region spanning amino acids 84–102 and another is found in the region spanning amino acids 143–168. In contrast, a major immunodominant eptiope of murine MBP is found in the region spanning amino acids 1–9 (Zamvil et al. Nature 324:258, 1986) and a major immunodominant epitope of rat MBP is found in the region spanning amino acids 68–88 (Burns, et al. J. Ex. Med. 169:27, 1989).
The use of antigens isolated from human CNS tissue as therapeutic agents is, however, undesirable. This is due not only to problems associated with purifying antigens from CNS tissue generally and the difficulty of obtaining human raw materials, but, more importantly, to the problem of eliminating the possibility of pathogenic contamination. A particularly intractable problem is the purification of CNS-derived proteins so as to eliminate possible contamination with the prion particles that transmit the spongiform encephalopathies Creutzfeldt-Jakob disease and kuru. The prion particles are resistant to any known means of sterilization that will not also destroy the proteins being purified.
A useful approach to obtaining human antigens that avoids these problems is the production of protein antigens using recombinant DNA technology, typically by preparing DNA molecules encoding the antigens and using the DNAs to drive expression of the antigens in non-human host cells. Oettinger et al. (1993) have prepared a recombinant DNA molecule comprising unmodified human sequences encoding the 18.5 kDa form of human MBP and used this DNA to express recombinant human 18.5 kDa MBP in Escherichia coli. 
T Cell Deletion
Alterations in the T cell repertoire occur naturally during T cell development. Only a small fraction of thymocytes (immature T cells) survive the development and selection events in the thymus that result in emigration of developing T cells to the peripheral circulation where they complete their maturation (von Boehmer, 1988; Marrack and Kappler, 1987). Experimental evidence strongly suggests that a large number of thymocytes that bear receptors for autoantigens are initially present in the thymus. During T cell development in the thymus, those cells reactive with self antigens are deleted (killed) as part of the normal developmental pathway. This intrathymic tolerization process is referred to as “thymic tolerance”.
Developing T cells do not encounter certain autoantigens in the thymus, but may encounter them as mature peripheral T cells. Tolerance to such autoantigens is normally produced outside the thymus, and is referred to as “peripheral tolerance”. Peripheral tolerance can occur by at least two mechanisms, one of which is a similar but distinct process to thymic tolerization that results in the deletion of those mature peripheral T cells that are specifically reactive with a previously unencountered autoantigen. In addition, T cells with certain specific reactivities can be induced to become inactive (anergic). Peripheral deletion and the induction of anergy are physiologic mechanisms that result in the development of “peripheral tolerance”. As a result of thymic and peripheral tolerization, mature T cells are normally tolerant to most autoantigens.
The mechanism by which tolerization via T cell deletion is generated has recently been shown to depend upon repeated exposure to an antigen under certain defined conditions. Specific T cell deletion can therefore be induced by the appropriate administration of exogenous compounds comprising the relevant epitopes. As only a limited number of autoantigens (notably comprising a much greater number of epitopes) are involved in the pathogenesis of any individual autoimmune disease, it is possible, when they are known, to administer the self epitopes targeted in a disease to sufferers in the form of one or more isolated autoantigen-derived compounds containing the epitopes involved in pathogenesis.
Apoptosis
The deletion of autoreactive T cells is an example of programmed cell death, which represents an important process in the regulation of many biological systems. Programmed cell death occurs by a mechanism referred to as apoptosis, in which cells respond to certain stimuli by undergoing a specific sequence of predetermined events that effectively constitute cellular suicide. Apoptosis clearly plays a large role in shaping and maintaining the T cell repertoire and contributes to the establishment of self-tolerance by actively eliminating cells expressing autoreactive TCRs.
It has recently been discovered that T cells are sensitive to apoptotic cell death induced by a variety of stimuli at multiple points in their lifespan (see, for example, Lenardo 1991. Nature 353, pp. 858–860; Boehme and Lenardo 1993. Eur J Immunol 23, pp. 1552–1560; Critchfield et al. 1994. Science 263, pp. 1139–1143). Positive selection factors are also believed to play a role in regulating the survival of specific T cell clones. The reduction or expansion of the number of individual T cells of a particular clone in an organism by these and other mechanisms serve to modulate the responsiveness of the organism's immune system to a particular antigen. It is now firmly established in several autoimmune disease models, as well as in certain viral infections, that apoptosis can be induced (upon exposure to antigen under certain defined conditions) in mature peripheral antigen-specific T lymphocytes as well as in immature thymocytes.
Apoptosis occurs in many biological systems (see, for example, Kerr et al. 1991; Lockshin and Zakeri, 1991; Cohen et al. 1992; Duvall and Wyllie, 1986; Cotter et al. 1990). A cell undergoing apoptosis undergoes a specific program of events—cellular and biochemical processes that depend upon active metabolism and contribute to the cell's self-destruction. In apoptotic T cells, the nucleus shrinks, the chromatin condenses, the genetic material (DNA) progressively degrades into small (nucleosomal repeat sized) fragments, there is cytoplasmic compaction, the cell membrane forms blebs, and the cell eventually collapses (Kawabe and Ochi, 1991; Smith et al. 1989). Cells cannot recover from apoptosis, it results in irreversible cell death (Kawabe and Ochi, 1991; Smith et al. 1989).
Recent reports have indicated a role for the TNF-related cytokine known as the FAS ligand and its receptor, CD95 (the FAS receptor), in the induction of apoptosis in T cells (Strasser, Nature 1995, 373:385; Dhein et al., Nature 1995, 373:438; Brunner et al., Nature 1995, 373:441; and Ju et al., Nature 1995, 373:444).
T cells that do not undergo apoptosis, but which have become activated, will carry out their “effector” functions by causing cytolysis, or by secreting lymphokines that cause B cell responses or other immune effects (Paul, 1989). These effector functions are the cause of tissue damage in autoimmune and other diseases.
Therapeutic Induction of Apoptosis
A powerful approach to avoiding or treating autoimmune diseases is to permanently eliminate by apoptosis only those T cells reactive with autoantigens targeted in the particular autoimmune disease being treated, while leaving the vast majority of the T cell repertoire intact. In vivo studies have demonstrated that EAE can be treated by administration of myelin antigens at a dose and interval effective to induce apoptosis of T cells reactive with the antigens (se, for example, Critchfield et al. 1994. Science 263, pp. 1139–1143).
This approach is described in U.S. patent application Ser. No. 07/751,090, which was abandoned in favor of continuing U.S. patent application Ser. No. 08/122,345, which issued as U.S. Pat. No. 6,083,503 in the name of Michael J. Lenardo, and entitled Interleukin-2 Stimulated T Lymphocyte Cell Death for the Treatment of Autoimmune Diseases, Allergic Responses, and Graft Rejection, and U.S. patent application Ser. No. 07/926,290, which was abandoned in favor of continuing U.S. patent application Ser. No. 08/348,286, which issued as U.S. Pat. No. 5,935,575, in the name of Michael J. Lenardo, and entitled Interleukin-4 Stimulated T Lymphocyte Cell Death for the Treatment of Allergic Disorders.
The accompanying figures, which are incorporated in and constitute part of the specification, illustrate certain aspects of the invention, and together with the description, serve to explain the principles of the invention. It is to be understood, of course, that both the figures and the description are explanatory only and are not restrictive of the invention.